1. Field of the Invention
The present invention-relates generally to blood coagulation testing, and more particularly to a test article comprising the reagents necessary for such an assay and methods which rely on the generation of a fluorescent or other visible signal from the test article.
Blood coagulation tests may be performed for a variety of purposes, including determination of bleeding susceptibility of patients undergoing surgery and monitoring of patients undergoing anti-coagulation therapy for the prevention of blood clots. A variety of coagulation tests are presently in use. One of the most popular is the "prothrombin time" (PT) test which relies on induction of the extrinsic coagulation pathway, and another is the "activated partial thromboplastin time" (APTT) test which relies on induction of the intrinsic coagulation pathway. Both the extrinsic and intrinsic coagulation pathways result in the production of thrombin, which is a proteolytic enzyme which catalyzes the conversion of fibrinogen to fibrin. Such conversion is an essential function in the clotting mechanism. Conveniently, thrombin production can be monitored by exposure of a patient blood sample to a synthetic thrombin substrate peptide, cleavably bound to a reporter molecule which is activated by thrombin cleavage. The reporter molecule produces an observable change, such as color production or fluorescence; and thrombin activity (which is a measure of the blood's coagulation capability) is assessed by optical means.
Heretofore, such blood coagulation tests have tended to be complex, with performance generally limited to clinical laboratories. While such centralized testing may be adequate for surgical patients, visiting a doctor's office or a clinic on a regular basis to monitor anti-coagulation therapy is less acceptable. Thus, the need for a convenient and practical home coagulation monitoring test is apparent.
The technical challenges in devising a coagulation test which is simple enough to be administered by the patients themselves, however, are substantial. The test must be extremely simple, low cost, robust, and must permit use with widely varying volumes of whole blood. Blood sampling in a home environment is generally limited to the "fingerstick" method (where a finger is pricked by a small needle mounted on a spring-loaded device), which produces relatively uncontrolled volumes of whole blood, generally being from 5 to 30 .mu.l. Additionally, it would be desirable if the blood coagulation test could be performed at room temperatures, eliminating the need for elaborate temperature control apparatus.
Successful home blood tests have been devised for other chemistries, such as cholesterol and glucose. Among the most suitable devices for home use are "test strips" comprising a layer of absorbent material having appropriate impregnated regions to perform a desired analysis. For example, a test strip for performing blood glucose analysis, available from Lifescan, Inc., Milpitas, Calif., relies on applying a drop of blood to a polyamide membrane having impregnated reagents which produce a chromogenic reaction in response to the glucose level in the applied blood. No equivalent test devices have been developed for measuring blood coagulation capability. While certain test articles for measuring coagulation have been proposed, even the most simple employ multiple-layers, where premeasured volumes of blood must be applied. These tests are generally performed in a clinical setting.
The nature of the blood coagulation pathways renders the performance of single layer test strip coagulation assays problematic. Blood coagulation involves a series of complex and poorly understood enzymatic reactions that are highly sensitive to surface interface effects. Additionally, blood contact with certain materials can inactivate the enzymes necessary for induction of the coagulation pathways. Thus, most previous coagulation assays have utilized containers with minimal surface area to reduce the likelihood of inadvertent activation or inhibition of the coagulation pathway. Test strip assays, in contrast, are generally performed in highly porous materials having a very large surface area. Thus, such test strip membranes would generally be contraindicated for use in blood coagulation assays.
For the above reasons, it would be desirable to provide simplified test articles and methods for measuring a patient's coagulation capability. The test articles and methods should be sufficiently simple and reliable to permit tests to be performed by untrained individuals, particularly by the patients themselves in a home setting. Preferably the tests should require only a single step, such as the application of a drop of blood onto a test article, followed by automated reading of the test results. The test should not be sensitive to variations in blood sample volume and should be capable of performance with minimal or no temperature control. In particular, it would be desirable to provide test articles and methods employing a porous membrane test strip, where the test strip has substantially no effect on the blood coagulation pathway being measured.
2. Description of the Background Art
Thrombin substrates suitable for use in coagulation assays are described in U.S. Pat. Nos. 3,884,896 and 4,640,893. A tableted form of thromboplastin having improved stability resulting from calcium separation is described in U.S. Pat. No. 4,755,461. A dry reagent consisting of thromboplastin and a thrombin substrate useful for liquid phase coagulation assays is described in U.S. Pat. No. 4,458,015.
An assay system for measuring glucose in blood, consisting of an inert porous matrix impregnated with reagents which produce a light-absorbing reaction, is described in U.S. Pat. No. 4,935,346. A blood sample is applied to one side of the matrix and reflectance measured on the opposite side. The membrane used in this system tends to rupture (lyse) red cells. This causes high levels of hemoglobin to permeate the interior of the membrane; and high levels of hemoglobin can be observed on the opposite side of the matrix. The '346 patent teaches that for analytes such as glucose, this effect may be overcome by using a particular chemistry that produces an optical signal with spectral properties that can be distinguished over the dominating spectra of this released hemoglobin, and reading the results with dual wavelength optics that correct for the hemoglobin spectra. Such an approach would not work for the very delicate, and hemoglobin sensitive, thrombin substrate coagulation chemistry. Another assay system suitable for measuring blood glucose, cholesterol, and urea is described in U.S. Pat. No. 4,774,192. The necessary reagents are impregnated in a structure, such as a porous membrane or bibulous film, which may be an asymmetric membrane. The '192 patent also suggests the use of flow control agents for controlling sample distribution in the structure.
U.S. Pat. Nos. 4,861,712, 4,910,510, and 5,059,525, describe multi-layer test articles suitable for monitoring blood coagulation. The '712 patent describes a complex structure consisting of a filamentous material, and a water soluble non-ionic polymer which coats and impregnates the filamentous material. This structure may contain thromboplastin and a detectable thrombin substrate, and is useful for monitoring a number of blood coagulation parameters, including prothrombin time.
The '525 patent describes a complex dry reagent for blood coagulation tests that contains a carrier material, and dried in it: a protease reactive to prothrombin (or Factors VII-X), a chromogenic protease substrate, a buffer, and a second coagulation factor. The inclusion of a protease into the dry reagent is useful for performing assays for coagulation factors, such as factor X, that must first be activated by proteolytic cleavage before their enzymatic activity can be measured.
U.S. Pat. No. 4,756,884, describes a capillary flow device for measuring blood characteristics, including prothrombin time.